Stable Pseudotyped Lentiviral Particles Using Syncytin Envelopes
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current lentiviral vector preparations face challenges such as low titers and limited transduction efficiency, especially when targeting primary cells which often require activation, leading to loss of naive status. Additionally, the stability and industrial scalability of lentiviral particles pseudotyped with certain envelope glycoproteins are issues.
Innovation Solution
The development of stable pseudotyped lentiviral particles using endogenous retroviral syncytins like HERV-W, HERV-FRD, and murine syncytin-A, which are highly fusogenic and can be frozen without significant loss of titer, improving transduction efficiency and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional lentiviral vectors are used to transduce primary cells, then transduction can be achieved, but activation of cells is required which causes loss of naive status
Solution Approach 1:
The patent changes the envelope glycoprotein parameter from conventional options (VSV-G, RD114TR) to endogenous retroviral syncytins (HERV-W, HERV-FRD, murine syncytin-A). This parameter change enables transduction of naive primary cells without activation, preserving their naive status while achieving efficient transduction. The syncytin-pseudotyped particles specifically recognize and bind to receptors on naive immune cells, allowing direct transduction without requiring cell activation.
2Quantity of substance
If lentiviral particles are concentrated during purification, then infectious titers are improved, but purification protocols are difficult to establish depending on envelope glycoproteins
Solution Approach 1:
The patent employs endogenous retroviral syncytins (HERV-W, HERV-FRD, murine syncytin-A) as envelope glycoproteins that provide universal transduction capability across multiple cell types including human and murine immune cells. This universality simplifies purification protocols because the syncytin-pseudotyped particles can be concentrated using standard methods without requiring cell-type-specific optimization, unlike conventional envelope glycoproteins that demand tailored purification approaches for each application.
3Productivity
If conventional envelope glycoproteins are used to pseudotype lentiviral vectors, then production can proceed, but stability during concentration and freezing is compromised with considerable titer reduction
Solution Approach 1:
The patent creates a composite pseudotype by combining the lentiviral particle core with endogenous retroviral syncytin envelope glycoproteins (HERV-W, HERV-FRD, or murine syncytin-A). This composite structure integrates the productive transduction capabilities of lentivectors with the exceptional stability of syncytins during concentration and freezing processes. The syncytin envelope forms a protective outer layer that maintains particle integrity and prevents aggregation, thereby preserving infectious titers through multiple freeze-thaw cycles and concentration steps.
4Adaptability or versatility
If various envelope glycoproteins are used to pseudotype lentiviral vectors, then specific cellular tropism can be conferred, but transduction efficiency on target cells such as CD34+ cells and primary blood cells remains variable
Solution Approach 1:
The patent optimizes the envelope glycoprotein parameter by selecting endogenous retroviral syncytins (HERV-W, HERV-FRD, murine syncytin-A) that naturally exhibit high affinity for receptors on immune cells including CD34+ hematopoietic stem/progenitor cells, T lymphocytes, B lymphocytes, and dendritic cells. This parameter change ensures consistently high transduction efficiency across diverse immune cell types while maintaining the ability to confer specific cellular tropism. The syncytins' natural receptor recognition properties provide reliable and reproducible transduction without the variability associated with conventional envelope glycoproteins.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These stable lentiviral particles demonstrate enhanced transduction efficiency for immune cells, including B cells, T cells, and dendritic cells, with minimal pre-activation requirements, and show detectable, stable, and well-tolerated gene transfer in vivo, particularly in spleen and bone marrow.
Implementation Method 1
Syncytins are endogenous retroviral virus (ERV syncytins) envelope glycoproteins which have fusogenic properties
Implementation Method 2
stable pseudotyped lentiviral particles pseudotyped with an endogenous retroviral syncytin which can be frozen
Data Source
Figure 1
Figure 2A~2D
Figure 3
AI summary
The present invention relates to a method for obtaining stable pseudotyped lentiviral particles including a heterologous gene of interest, comprising the following steps: a) transfecting at least one plasmid in appropriate cell lines, wherein said at least one plasmid comprises the gene of interest, the rev, gag and pol genes, and a sequence coding for an ERV syncytin, wherein the rev, gag and pol genes are retroviral genes; b) incubating the transfected cells obtained in a), so that they produce the stable pseudotyped lentiviral particles in the supernatant; and c) harvesting and concentrating the stable lentiviral particles obtained in b). The present invention also relates to a method to transduce immune cells using lentiviral vectors pseudotyped with an ERV syncytin glycoprotein. The method can be performed on non-stimulated blood cells or on cells stimulated briefly with IL7, and the cells can be expanded. The stable pseudotyped lentiviral particles obtained are particularly useful in gene therapy.